The presence of thyroid stem/progenitor cells has been demonstrated in recent years, however due to no specific cell surface markers known, and small organ size and lack of tissues, the progress has been slow. We previously isolated Hoechst dye-resistant side population (SP) cells from mouse thyroids by fluorescence-activated cell sorting (FACS). Thyroid SP cells demonstrated stem/progenitor characteristics. Using these mouse thyroid-derived SP cells, a cell line named SPTL (side population cells-derived thyroid cell line) was established. This continuous source allows the study and characterization of thyroid stem/progenitor cells. Characterization of SPTL cells were carried out by using in vitro 2- and 3-dimensional cultures and in vivo reconstitution of mice, in conjunction with qRT-PCR, western blotting, immunohisto(cyto)chemistry and/or immunofluorescence, and RNA-seq analysis. Under low serum culturing conditions, SPTL cells expressed the thyroid differentiation marker NKX2-1, a transcription factor critical for thyroid differentiation and function, while no expression of other thyroid differentiation marker genes was observed, which included PAX8, another critical transcription factor for thyroid differentiation and function. It was reported that the expression of both NKX2-1 and PAX8 is essential for differentiation of ES cells to become functional thyroid. SPTL cells formed follicle-like structures in 3-dimensional matrigel cultures, which did not express thyroid differentiation marker genes. In mouse models of orthotopic and intravenous injection, the latter following partial thyroidectomy as the model for thyroid injury, a few SPTL cells were found in part of the follicles, most of which expressed NKX2-1. Based on these results, we hypothesized that NKX2-1 is required for thyroid follicle formation, while other thyroid transcription factors such as PAX8 are required for the follicle to become functional. This hypothesis needs to be proven in future studies. Further, RNA-seq analysis demonstrated that SPTL cells highly express genes involved in epithelial-mesenchymal transition, and exhibit a gene expression pattern similar to anaplastic thyroid carcinoma. Thus, SPTL cells may be characterized as partially thyroid-specified progenitor-like cells, with the capacity to differentiate into thyroid to a limited degree. SPTL cells may provide an excellent tool to study stem cells, including cancer stem cells of the thyroid.